Antiviral or antifungal composition comprising an extract of pomegranate rind or other plants and method of use

ABSTRACT

Antiviral and antifungal compositions comprising a mixture of a ferrous salt and a plant extract of pomegranate rind, Viburnum plicatum leaves or flowers, tea leaves, or maple leaves in an aqueous solution are disclosed. The compositions are mainly used to prevent the growth of, or kill, viruses or fungi on surfaces. The compositions do not substantially affect bacterial viability as measured by colony forming ability.

Bacteriophage represent a diverse group of viruses that exert bothpositive and negative effects in microbiology. For example, in the dairyindustry bacteriophages of the lactic acid bacteria represent a majorsource of starter culture failure with consequent poor-quality milkfermentations. A number of measures to prevent phage infection,including implementation of hygienic procedures, rotation of startercultures, isolation of resistant mutants and formulation of media tosuppress phage proliferation have been instituted with various degreesof success 1-4!. By contrast, coliphage have proven to be valuablemonitors of faecal contamination in ground water and treated drinkingwater 5! and are effective viral models in studies of water quality andvirucidal activity 6, 7!. Recent developments with geneticallyrecombinant bacteriophage, containing either the bacterial luciferaselux genes or the ice nucleation gene ina, have established an additionalvalue for phage in the rapid detection of bacteria in food andenvironmental samples 8-12!. In the above examples, the eventualdestruction of bacteriophage is important either from the perspective ofmaintaining effective fermentation or from the viewpoint of goodmicrobiological practice in the disposal of contaminated material. Atpresent, the methods available for bacteriophage destruction such asheat and chemical disinfection 13, 14!, have a significant impact on theviability and survival of associated bacteria. In the case of asterilisation regime this is of little significance but the ability tocombat bacteriophage in industrial environments would benefit from anenvironmentally benign procedure that could differentially destroy thevirus without damaging metabolically-active bacterial cells. One exampleis in the dairy industry as mentioned above. Another concerns sprayingplants which have symbiotic bacteria to control bacteriophage infection.

There are also many situations where it would be useful to be able tocombat fungus without at the same time damaging metabolically-activecells. Plant extracts have been used against disease development inbanana by fungi 15!.

This invention provides an antiviral or antifungal compositioncomprising an effective concentration of a mixture of a ferrous salt andan extract of a plant selected from pomegranate rind, Viburnum plicatumleaves or flowers, tea leaves and maple leaves.

An antiviral composition is one which combats, e.g. by preventing growthor preferably killing, virus such as bacteriophage. An antifungalcomposition is one which combats, e.g. by preventing growth andpreferably by killing fungus and destroying its spores. Preferredcompositions are antiviral or antifungal, but without at the same timesubstantially damaging metabolically active bacterial or other cellswith which the virus or fungus is associated. Preferably the compositionis an aqueous solution, i.e. one in which water is the sole or the mainconstituent solvent.

The composition comprises a ferrous salt, which may conveniently beferrous sulphate. The nature of the anion is however not critical,provided that the salt is non-toxic to bacterial and other cells underthe intended conditions of use and is water-soluble. A preferred ferroussalt concentration range is 0.1 mM to 0.1M, particularly from 1 to 20mM.

The composition also contains an extract of a plant. These plantextracts may conveniently be prepared by boiling the comminuted plantpart with water or other solvent. The resulting extract may befractionated. It is probable that the extract contains one or moreactive components. Although such active components have remainedrefractive to purification, there is a uniform consistency in theextracted activity from different sources of the plant parts and fromparts obtained at different times of the year. The inventors haveexamined many different plants and have identified the following four asactive:

Pomegranate rind. Whole pomegranates can be comminuted and used, but theactivity resides in the rind.

Viburnum plicatum leaves or flowers.

Tea leaves. These may be dried or green. Other parts of the tea plantCamellia sinensis may be used.

Maple leaves e.g. UK Acer pseudoylatanus or Canadian maple leaves, ormore generally leaves or flowers of any part of the genus Acer.

The plant extract may be used as is, or diluted as appropriate, e.g. bya factor of up to 100. Effective compositions generally contain 10-90%by volume of the ferrous salt solution together with correspondingly90-10% by volume of the concentrated or diluted plant extract. Thecomposition should preferably be stored in the dark.

The invention also includes solid or liquid concentrates which ondilution with water gives compositions as described.

The invention also includes a method of controlling virus or fungus,which method comprises contacting the virus or fungus with an effectiveconcentration of a ferrous salt and an effective concentration of anextract of a plant selected from pomegranate rind, Viburnum plicatumleaves or flowers, tea leaves and maple leaves. These two components canbe used in sequence in either order. Preferably, however they are usedmixed together as a composition as described above.

The virus or fungus to be controlled may be contacted with, e.g.immersed in, the composition, typically for a few seconds or minutes.Where the virus or fungus is on a surface, the composition may beapplied to the surface, e.g. by spraying or wiping.

The surface may be for example a work surface or a vessel or utensilused in a hospital or kitchen or an industrial environment, or anexternal surface of a mammal e.g. a human or a plant. Or a solutioncontaining virus or fungus may be mixed with a composition as defined.

As described in the examples below, ferrous sulphate in combination withselected plant extracts effects complete destruction of a broad range ofbacteriophage infecting diverse bacterial genera. In assaysincorporating both bacteriophage and bacteria at 10¹² and 10⁹ /mlrespectively, the bacteriophage are entirely destroyed within twominutes without affecting bacterial viability as measured by colonyforming ability.

When used alone, ferrous sulphate has virucidal activity against phages,and also bactericidal activity against some bacteria. This invention isbased on the observation that ferrous sulphate, either alone or incombination with certain plant extracts, offers a potent broad spectrumvirucidal activity. Since the activity of relatively low levels offerrous sulphate can be further potentiated by the addition of traceamounts of hydrogen peroxide (data not shown) it is likely that themechanism of action involves, at least in part, a free radical system. Amechanism similar to that operating in the phagolysozome and defined bythe Modified Haber-Weiss Reaction 17!is proposed.

    Fe.sup.2+ +H.sub.2 O.sub.2 →Fe.sup.3+ +OH.sup.- +.OH

While resistance of bacterial cells may be effected through free radicalscavenging and repair systems, it is clear that the plant extracts alsohave a role and that remains to be elucidated.

Materials and Method

Bacteria and bacteriophage strains

One Gram-positive Staphylococcus aureus NCIMB 8588 and two Gram-negativeSalmonella typhimurium LT2 and Pseudomonas aeruginosa NCIMB 10548bacteria were used. The bacteriophage with specificity for the abovebacteria were NCIMB 9563 for Staph.aureus, Felix 01 for S.typhimurium16!(obtained from Amersham International plc., Amersham, HP7 9NA, UK)and NCIMB 10116 and 10884 for Ps.aeruainosa. (All these bacteria andbacteriophage are available to the public.)

The bacterial cells were maintained on Tryptose Phosphate Broth (TPB;Oxoid) supplemented with 1% agar (TPA) and stored at 4° C. with monthlysubculture. When required, cells were resuscitated in 10 ml TPB (18h,37° C.) or an orbital shaker operating at 240 rpm. Appropriate bacterialdilutions were made in Lambda buffer (6 mM Tris, 10 mM MgSO₄.7H₂ O, 50μg/ml gelatin; pH 7.2). After treatment bacterial survival wasdetermined by colony forming units (cfu) on TPA 18!.

Phage stocks were developed on their appropriate host strains by a platelysis procedure essentially equivalent to growing bacteriophageLambda-derived vectors 19!. Typical phage titres of 10¹² /ml wereobtained. Phage stocks were maintained in Lambda buffer at 4° C. andstocks retained a constant titre for several months.

Preparation of plant extracts

Pomegranate rind, Viburnum plicatum leaves or flowers, maple leaves andcommercial tea leaves were blended in distilled water (25% w/v) andboiled for 10 min. After centrifugation (20,000 ×g, 4° C., 30 min),supernatants were autoclaved (121° C., 15 min), cooled and stored at-20° C. A further purification of the pomegranate extract to a molecularweight cut-off of 10,000 Da was achieved by membrane ultra filtrationand the filtrate stored as above.

EXAMPLE 1

Preparation of Composition A

a. Preparation of 4.3 mM FeSO₄.7H₂ O in Lambda-buffer

First freshly prepare stock solution (0.53%) of FeSO₄.7H₂ O (0.053 gmferrous sulphate in 10 ml Lambda-buffer). After sterilisation bymembrane filtration (0.45 μm, Whatman) prepare the final ferroussulphate concentration of 4.3 mM by transferring 4.1 ml of the ferrousstock solution to a sterile test tube containing 14 ml of Lambda-buffer.

b. Preparation of 13% PRE (pomegranate rind extract)

Mix 1.3 ml of stock solution of PRE (25% w/v) with 8.7 ml ofLambda-buffer.

Composition A was prepared 1-2 min prior to use by mixing 16.74 ml of4.3 mM FeSO₄.7H₂ O (a; yellow) with 8.265 ml of 13% PRE (b; yellow).After about 30 sec the colour of the mixture (a and b) changed greenishthen to black. These mixtures of ferrous sulphate and PRE (a and b)should be protected from light.

EXAMPLE 2

Virucidal Assay

Plant extracts were diluted 1:8 in Lambda buffer immediately prior touse and 300 μl of this diluted extract added to 700 μl of freshlyprepared ferrous sulphate solution (4.3 mM FeSO₄.7H₂ O; pH 6.5); thesemixtures should be protected from light. Bacteriophage (20 μl at 10¹²pfu/ml) or 20 μl of an appropriate dilution of bacteria (10⁹ cfu/ml)were placed in a sterile Eppendorf micro-centrifuge tube and 144 μl ofthe FeSO₄ solution or PRE solution or composition A (Example 1) or ofthe above plant extract/FeSO₄ mixture added. After exposure of thebacteriophage or bacteria for 2 min at room temperature the activity ofthe mixture was neutralised by adding an equal volume of 2t (v/v) Tween80 in Lambda buffer. The number of bacteriophage or bacteria survivingthe above protocol were measured by plaque forming units (pfu) or colonyforming units respectively.

The results are set out in the following Tables 1 and 2. Table 1indicates that pomegranate rind extract alone has a slight virucidalactivity against Pseudomonas phage but in combination with FeSO₄ thereis a profound synergy. Eleven log reductions in plaque forming abilityare obtained within 2 min. A similar synergy of virucidal action wasachieved with other plant extracts, leaves and flowers of Viburnumplicatum, maple leaves and tea leaves (Table 2).

Our particular interest in the above studies was to select agents withmaximal virucidal activity whilst having little effect on bacteria. Inthis regard FeSO₄, either alone or in combination with pomegranate rindextract, appears singularly successful. Table 1 shows that for eachbacterium and phage combination tested there is a simple treatment thatcan eliminate bacteriophage activity without affecting bacterialvisibility. The plant extracts appear to serve a dual role in theirinteraction with FeSO₄. In the case of Pseudomonas phage they promotevirucidal activity while for the Salmonella phage, which is completelyinactivated by FeSO₄ alone, the pomegranate rind extract appears toprovide a significant protection to the bacterium.

                                      TABLE 1                                     __________________________________________________________________________    The survival of different bacteria and                                        bacteriophage species in 4.3 mM FeSO.sub.4.7 H.sub.2 O, 13% PRE               (pomegranate rind extract; 20 min at 37° C.) and                       Composition A (PRE + 4.3 mM FeSO.sub.4.7 H.sub.2 O; 2 min at room             temperature) prepared in Lambda buffer.                                                     Lambda                                                                             13%  4.3 mM                                                Microorganisms                                                                              Buffer                                                                             PRE  FeSO.sub.4.7 H.sub.2 O                                                               Composition A*                                 __________________________________________________________________________    Bacteriophage species (pfu/ml):                                               Staphylococcus NCIMB 9563                                                                   7 × 10.sup.11                                                                2 × 10.sup.7                                                                 Nil    Nil                                            Salmonella Felix 01                                                                         3 × 10.sup.12                                                                3 × 10.sup.12                                                                Nil    Nil                                            Pseudamonas NCIMB 10884                                                                     5 × 10.sup.11                                                                5 × 10.sup.9                                                                 5 × 10.sup.11                                                                  Nil                                            Pseudamonas NCIMB 10116                                                                     5 × 10.sup.11                                                                5 × 10.sup.8                                                                 5 × 10.sup.11                                                                  Nil                                            Bacteria species (cfu/ml):                                                    Staph.aureus NCIMB 8588                                                                     3 × 10.sup.9                                                                 2 × 10.sup.6                                                                 3 × 10.sup.9                                                                   5 × 10.sup.6                             S.typhimurium LT2                                                                           2 × 10.sup.9                                                                 2 × 10.sup.9                                                                 Nil    2 × 10.sup.9                             Ps.aeruginosa NCIMB 10548                                                                   4 × 10.sup.9                                                                 4 × 10.sup.9                                                                 1 × 10.sup.7                                                                   4 × 10.sup.9                             __________________________________________________________________________     * Escherichia coli phage M13mp18 was killed by Composition A, whilst E.       coli JM101 was unaffected.                                               

                  TABLE 2                                                         ______________________________________                                        Effect of 25% solutions of different plant                                    extracts in the presence of 4.3 mMol ferrous sulphate                         on the inactivation within 3 minutes of Ps.aeruginosa                         bacteriophage NCIMB 10116.                                                    25% Plant extract solution (% v/v)                                                                 PFU/ml                                                   ______________________________________                                        Lambda Buffer (control)                                                                            1.2 × 10.sup.10                                    Commercial tea leaves                                                                              Nil                                                      Viburnum plicatum leaves or flowers                                                                Nil                                                      Maple leaves         Nil                                                      ______________________________________                                    

An antiserum was raised in rabbits against the Pseudomonas phage. Aclassic precipitin band was obtained with a double-immunodiffusion assayof phage antigen against this antiserum. No such precipitin band wasobtained, however, when the phage had been treated with the virucidalagent, nor if the antiserum had been raised against treated phage.

Treated phage were further examined by electron microscopy. Norecognisable phage morphological structures remained in any of thetwenty fields examined. The result of the further tests are consistent,therefore, with a complete destruction of the bacteriophage structure bythe virucidal composition, to an extent that antigenic determinants arelost.

EXAMPLE 3

Antifungal Test

Apple leaves infected with powdery mildew were immersed in Composition A(Example 1) for about 60 seconds or until the leaves were completelywetted. The leaves were then observed daily. The powdery mildew wascompletely inhibited within 24 hours and no mildew lesions were presenton the treated apple leaves as they hardened off.

A further microscopic test of treated powdery mildew spores andmycellium with Composition A showed that both spores and mycellium weredestroyed after 1 hour at room temperature. Composition A neutralised by2%. TW80 was not effective however.

EXAMPLE 4

Composition A was tested under the conditions described in Example 2 foractivity against poliovirus, herpes simplex virus type 1. (HSV-1) andhuman immunodeficiency virus type 1. 20 μl of an appropriate dilution ofvirus was mixed with 144 μl of composition A. After exposure, thevirucide was neutralised by addition of a 2% v/v solution of Tween 80 inlambda buffer. In cell culture assays using HT-29 cells (forpoliovirus), MRC-5 cells for HSV-1 and IIUT-78 cells for HIV-1 there wascomplete reduction of infectivity of initial inocula of 1-4 ×10⁶ virus.There was no apparent cytopathic effect on MRC-5 cells or HT-29 cells.

REFERENCES

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3. Thunell, R., Sandine, W. E. and Bodyfelt, F. (1981) Phageinsensitive, multiple-strain starter approach to cheddar cheese. J.Dairy Sci. 64, 2270-2277.

4. Sing. W. D. and Klaenhammer, T. R. (1993) A strategy for rotation ofdifferent bacteriophage defenses in a lactococcal single-strain starterculture system. Appl. Environ. Microbiol. 59, 365-372.

5. Dutka, B. J., Palmateer, G. A., Meissner, S. M., Janzen, E. M. andSakellaris, M. (1990) The presence of bacterial virus in groundwater andtreated drinking water. Environ. Pollut. 63, 293-298.

6. Jassim, S. A. A., Ellison, A., Denyer, S. P. and Stewart, G. S. A. B.(1990). In vivo bioluminescence: a cellular reporter fo research andindustry. J. Biolumin. Chemilumin. 5, 115-122.

7. Mesquita, M. M. F. D. (1990) Bacteriophages as viral models instudies of water and shellfish quality. Rev. Bras. Biol. 49, 923-932.

8. Ulitzur, S. and Kuhn, J. (1987) Introduction of lux genes intobacteria, a new approach for specific determination of bacteria andtheir antibiotic susceptibility. In Bioluminescence andChemiluminescence New Perspectives, (Schlomerich, J., Andereesen, R.,Kapp, A., Ernst, M. and Woods, W. G. Eds), pp. 463-472. Bristol:Wiley.

9. Stewart, G. S. A. B., Smith, A. T. and Denyer, S. P. (1989) Geneticengineering of bioluminescent bacteria. Food Sci. Technol. Today. 3,19-22.

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11. Kodikara, C. P., Crew, H. H. and Stewart, G. S. A. B. (1991) Nearon-line detection of enteric bacteria using lux recombinanatbacteriophage, FEMS Microbiol. Letts. 83, 261-266.

12. Turpin, P. E., Maycroft, K. A., Bedford, J., Rowlands, C. L. andWellington, E. M. H. (1993) A rapid luminescent-phage based MPN metahodfor the enumeration of Salmonella typhimurium in environmental samples.Letts. Appl. Microbiol. 16, 24-27.

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We claim:
 1. An antiviral or antifungal composition for treatingsurfaces, comprising an effective concentration of a mixture of aferrous salt and an extract of pomegranate rind, wherein saidcomposition does not substantially affect bacterial viability asmeasured by colony forming ability.
 2. The composition as claimed inclaim 1, which is an aqueous solution.
 3. The composition as claimed inclaim 2, wherein the ferrous salt concentration is 0.1 mM to 0.1M. 4.The composition as claimed in claim 2 or 3, wherein the pomegranate rindextract is used at a dilution of 1 to 100 times.
 5. The composition asclaimed in claim 2 or 3, wherein the ferrous salt is ferrous sulphateused at a concentration of 1-20 mM.
 6. A solid or liquid concentratewhich on dilution with water gives a composition according to claim 2 or3.
 7. A method of preventing growth of, or killing, viruses or fungi ona surface, which method comprises contacting the virus or fungus with acomposition comprising an effective concentration of a mixture of aferrous salt and an extract of pomegranate rind, wherein saidcomposition does not substantially affect bacterial viability asmeasured by colony forming ability.
 8. The method as claimed in claim 7,wherein the ferrous salt is used as a 0.1 mM to 0.1M aqueous solution.9. The composition as claimed in claim 4, wherein the ferrous salt isferrous sulphate used at a concentration of 1-20 mM.
 10. A solid orliquid concentrate which on dilution with water gives a compositionaccording to claim
 4. 11. A solid or liquid concentrate which ondilution with water gives a composition according to claim
 5. 12. Asolid or liquid concentrate which on dilution with water gives acomposition according to claim
 9. 13. The method as claimed in claim 7wherein the virus or fungus is contacted with an aqueous compositioncomprising a mixture of the ferrous salt and the plant pomegranate rindextract.